The family Flaviviridae includes the prototype yellow fever virus (YFV), the four serotypes of dengue virus (DENY-1, DENV-2, DENV-3, and DENV-4), Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV), West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), and about 70 other disease causing viruses. Flaviviruses are small, enveloped viruses containing a single, positive-strand RNA genome. Ten gene products are encoded by a single open reading frame and are translated as a polyprotein organized in the order: capsid (C), “preMembrane” (prM, which is processed to “Membrane” (M) just prior to virion release from the cell), “envelope” (E), followed by non-structural (NS) proteins NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5 (reviewed in Chambers, T. J. et al., Annual Rev Microbiol (1990) 44:649-688; Henchal, E. A. and Putnak, J. R., Clin Microbiol Rev. (1990) 3:376-396). Individual flaviviral proteins are then produced through precise processing events mediated by host as well as virally encoded proteases.
The envelope of flaviviruses is derived from the host cell membrane and contains the virally-encoded membrane-anchored membrane (M) and envelope (E) glycoproteins. The E glycoprotein is the largest viral structural protein and contains functional domains responsible for cell surface attachment and intra-endosomal fusion activities. It is also a major target of the host immune system, inducing the production of virus neutralizing antibodies, which are associated with protective immunity.
West Nile virus has become an emerging infectious disease in the United States. The virus infects birds, which serve as the natural reservoir for the virus, in addition to humans and horses, which are incidental hosts. It is an arthropod-borne virus transmitted by over 42 species of mosquitoes from various genera including the genus Culex. The first documented case of WNV was found in the West Nile region of Uganda in 1937 (Smithburn et al., Am J Trop Med Hyg (1940) 20:471-492). It has since spread through the Middle East, Oceania, parts of Europe and Asia, and has recently emerged in the Americas. Since the first case of human infection in the U.S. was documented in New York City in 1999, the virus rapidly spread throughout the East coast of the U.S. and has spread west across the continent. It has now been found in bird populations in all 48 continental states. Human cases of WN disease have been documented in 47 of the 50 states, with only Alaska, Hawaii and Maine having no reported human cases (MMWR, 2008, 57(26):720-23).
The majority of individuals infected with WNV experience flu-like symptoms. However, a number of infected individuals will develop severe disease which carries a case-fatality rate of 3-15% and is highest among the elderly. In addition, in a high percentage of the non-fatal cases, permanent neurological disabilities result. In 2003, of 9,862 symptomatic infected individuals, 2,866 (29%) had neuroinvasive disease (defined as West Nile meningitis, encephalitis and myelitis) and 264 died from the disease. Neuroinvasive complications rose to 36% in 2004 (MMWR, vol. 53 Nov. 19, 2004). Recent studies have shown that recovery from viral infection requires significantly more time than originally thought. One study has concluded that the median recovery time was 60 days (Comment, Ann Inter. Med. (2004), 141:153) while another documented that only 37% of patients recovered completely after one year (Klee et al., Emerg. Inf. Dis. (2004) 10:1405-1411). The neurological damage done by the virus is slow to heal and, in some cases, is permanent. In recent years, some individuals have suffered from polio-like symptoms of acute flaccid paralysis. The clinical findings are significantly worse in elderly patients. In a study of a recent outbreak of WNV infections in Israel, within the study group of 233 hospitalized patients, there was an overall case fatality rate of 14%. However, among patients aged 70 or older, the case fatality rate was 29% (Chowers et al., Emerg. Inf Dis. (2001) 7:675-78). Similar findings were also reported from recent epidemics in Romania (Tsai et al., Lancet (1998) 352:767-771) and Russia (Platonov et al., Emerg. Inf. Dis. (2001) 7:128-32). Thus, there is significant morbidity and mortality associated with WN disease, especially among the elderly/immunosenescent, immunocompromised, and immunosuppressed populations.
The WNV envelope protein shares significant homology with the envelope proteins of other flaviviruses, particularly those in the Japanese encephalitis (JE) serocomplex: JEV, St. Louis encephalitis (SLEV), and Murray Valley encephalitis (MVEV) viruses. Antibodies directed against particular epitopes contained within the envelope protein are capable of viral neutralization, i.e., the inhibition of virus infection of susceptible cells in vitro. Neutralizing antibody epitopes have been mapped to all three domains of the E glycoprotein of flaviviruses, including WNV (Diamond et al., Immunol. Rev. (2008) 225:212-25). A high titer of viral neutralizing antibodies is generally accepted as the best in vitro correlate of in vivo protection against flavivirus infection and the resultant disease (Markoff Vaccine (2000) 18:26-32; Ben-Nathan et al., J Inf. Diseases (2003) 188:5-12; Kreil et al., J. Virol. (1998) 72:3076-3081; Beasley et al., Vaccine (2004) 22:3722-26). Therefore, a vaccine that induces high titer WNV neutralizing antibody responses will likely protect vaccines against disease induced by WNV.
To date the development of flavivirus vaccines has met with mixed success. There are four basic approaches that have been implemented in an effort to produce vaccine candidates to protect against disease causes by flaviviruses. The four methods are live-attenuated virus, inactivated whole virus, recombinant subunit protein, and DNA. The live-attenuated virus vaccine developed for YFV has been available for many decades and demonstrates the utility of this approach. The use of inactivated whole virus vaccines has been demonstrated for TBEV and JEV with registered products available for both of these disease targets based on this approach.
As described above, there has been success in developing vaccines for YFV, JEV, and TBEV. However, the use of live-attenuated virus and inactivated virus methods to develop vaccines for other flaviviruses has met significant challenges. For example, a significant amount of effort has been invested in developing candidate live-attenuated dengue vaccine strains; however, many of the strains tested have proven unsatisfactory (see, e.g., Eckels, K. H. et al., Am. J Trop. Med. Hyg. (1984) 33:684-689; Bancroft, W. H. et al., Vaccine (1984) 149:1005-1010; McKee, K. T., et al., Am. J Trop. Med. Hyg. (1987) 36:435-442). Despite these initial unsatisfactory results, efforts to develop and test dengue live-attenuated candidate vaccine strains continue (Reviewed in Am. J. Trop. Med. Hyg. (2003) 69:1-60). No significant efforts to develop a WNV vaccine utilizing traditional live-attenuated methods have been made.
As an alternative to traditional live-attenuated methods to develop flavivirus vaccines, recombinant chimeric methods have been utilized. This method utilizes a known live-attenuated flavivirus strain as a base and the appropriate genes (prM and E for flaviviruses) from a related virus of interest are substituted for the equivalent genes of the base virus. One approach that has been used for WNV and DENY vaccine development is use of an intertypic chimeric based on an attenuated DENV-4 strain (Bray, M: et al., J. Virol. (1996) 70:4162-4166; Chen, W., et at, J. Virol. (1995) 69:5186-5190; Bray, M. and Lai, C.-J., Proc. Natl. Acad. Sci. USA (1991) 88:10342-10346; Lai, C. J. et at, Clin. Diagn. Virol. (1998) 10:173-179). Another approach has been the use of the YFV 17D attenuated strain as a base to develop recombinant chimeric vaccines for JEV, DENY, and WNV (Lai, C. J. and Monath T. P. Adv Virus Res (2003) 61:469-509; Monath et al. Proc. Natl. Acad. Sci. USA (2006) 103:6694). While the use of live-attenuated chimeric methods has advantages over traditional live-attenuated methods, the chimeric methods are still plagued by difficulties faced in developing properly attenuated strains and in the case of a DENY vaccine achieving balanced, tetravalent responses against the four dengue viruses. Furthermore, live-attenuated approaches may not be appropriate for vaccines targeting encephalitic diseases due to an elevated risk factor or for target populations with compromised immune systems. Both of these factors are applicable to WNV vaccine development.
Currently there are commercially available vaccines produced for JE and TBE utilizing the whole inactivated virus methods. As with live-attenuated virus methods, the use of inactivated virus methods for certain flaviviruses has not guaranteed success with other flaviviruses. For example, efforts to develop inactivated DENV or WNV vaccines have met with limited success. This method is limited by the ability to obtain adequate viral yields from cell culture systems. Virus yields from insect cells such as C6/36 cells are generally in the range of 104 to 105 pfu/ml, well below the levels necessary to generate a cost-effective inactivated virus vaccine. Yields from mammalian cells including LLC-MK2 and Vero cells are higher, but the peak yields, approximately 106 pfu/ml from a unique Vero cell line, are still lower than necessary to achieve a cost-effective vaccine product.
The use of naked DNA methods has also been evaluated in an effort to develop non-replicating flavivirus vaccines for DENV, JEV, TBEV and WNV (Reviewed in Putnak, R. et al. (2003) Adv. Virus Res. 61:445-68). The DNA method offers advantages in ease of production, use of defined sequences, potential to elicit both humoral and cellular immunity due to the expression of virus antigens in vivo. Despite these advantages, the ability to induce consistent and robust immune responses in humans, particularly antibody responses, continues to be a major hurdle to this approach. Additionally, DNA vaccines face additional regulatory scrutiny due to concerns about integration of plasmid sequences in the host genome and the potential of generating auto-antibodies to double stranded DNA. To date no DNA vaccine has been approved for human use and it is not clear that this approach will ever be deemed appropriate for a prophylactic human vaccine.
The use of recombinant subunit proteins for flavivirus vaccine development is another example of a non-replicating virus approach. This approach offers advantages in production of well defined products and the potential to elicit specific immune responses. While the potential to generate relevant and robust immune responses exist, there are challenges associated with use of recombinant subunit protein vaccines. This is due to both the quality of the proteins (native-like structure) and the need for adjuvants in eliciting the desired immune responses. Recombinant subunit protein vaccines have a long history of safety and protective efficacy, illustrated most effectively by the recombinant subunit Hepatitis B vaccines (e.g. Engerix B® and Recombivax HB®), and more recently by the human papilloma virus vaccines (e.g. Gardasil® and Cervarix®). The fact that there is no replicating virus present at any time during production, helps assure that there is very limited risk associated with the administration of the subunit vaccine to healthy or immunocompromised individuals in a prophylactic setting. Moreover, the Hepatitis B and human papillomavirus vaccines have been shown to be highly immunogenic and efficacious.
The expression of recombinant flavivirus proteins has focused on the structural proteins C, prM and E and the non-structural protein NS1. The E protein has been the subject of most efforts as this protein is exposed on the surface of the virus, is involved in important biological aspects of the virus life cycle (e.g. binding to receptors and mediating fusion), and is the target of neutralizing antibodies in infected hosts (Chambers, supra; Mason, P. W., J. Gen Virol (1989) 70:2037-2048). Furthermore, monoclonal antibodies directed against purified flavivirus E proteins are neutralizing in vitro and some have been shown to confer passive protection in vivo (Henchal, E. A. et al., Am. J. Trop. Med. Hyg. (1985) 34:162-169; Heinz, F. X. et al., Virology (1983) 130:485-501; Kimura-Kiroda, J. and Yasui, K., J Immunol. (1988) 141:3606-3610; Trirawatanapong, T. et al., Gene (1992) 116:139-150; Money, J. D. et al., J. Inf. Dis (2006) 194:1300-8).
A variety of expression systems such as E. coli, yeast, and baculovirus have been utilized for the production of recombinant flavivirus proteins for use in vaccines. These attempts have been plagued by low yields, improper processing of the flavivirus proteins, and moderate to poor immunogenicity (Eckels, K H and Putnak, R, Adv. Virus Res. (2003) 61:395-418). Work at Hawaii Biotech, Inc. (HBI) on expression of recombinant E subunit proteins has established the need to maintain the native-like structure of the E protein in order for the recombinant proteins to serve as potent immunogens. The ability to produce recombinant E proteins with native-like structure is highly dependent on the expression system utilized. U.S. Pat. No. 6,165,477 discloses the process for expression of DENY E protein subunits in yeast cells. The E subunits expressed in yeast cells demonstrated improved structure over bacterial systems, but still faced problems with hyper-glycosylation, yields, and product uniformity.
In more recent studies, it has been established that the use of stably transformed insect cells to express truncated forms of the E protein results in uniform products that maintain native-like structure as determined by X-ray crystallography (Modis, Y. et al, Proc. Natl. Acad. Sci. USA (2003) 100:6986-91; Modis, Y. et al, Nature (2004) 427:313-19; and Zhang, Y. et al, Structure (2004) 12:1607-18). The use of the stably transformed insect cell system has resulted in successful expression of truncated recombinant E subunit proteins from DENV-1, -2, -3, -4, JEV, TBEV and WNV. U.S. Pat. No. 6,136,561 discloses the process for expression of DENV, JEV, TBEV and YFV recombinant E subunit proteins in stably transformed insect cells. U.S. Pat. No. 6,432,411 discloses the utility of flavivirus E recombinant subunit proteins expressed in stably transformed insect cells as candidate vaccines when combined with saponin containing ISCOM-like structures. Preclinical evaluation of the truncated recombinant WNV envelope subunit protein (WN-80E) and non-structural 1 protein (WN-NS1) produced by stably transformed insect cell lines has been recently reported (Lieberman et al, Vaccine (2008) 25:414-423; Watts et al, Vaccine (2007) 25:2913-2918; Siirin et al., Am. J Trop. Med. Hyg. (2008) 79:955-962). In these reports combinations of E and NS1 formulated with saponin containing adjuvants are evaluated in mice and hamster models to evaluate protective efficacy. These patents and publications demonstrate the utility of the flavivirus recombinant subunit proteins expressed from stably transformed insect cells when combined with saponin containing adjuvants to generate appropriate antibody responses in animal models. U.S. Pat. No. 6,432,411 along with Lieberman et al. 2007 (supra) and Watts et al. 2007 (supra) also demonstrate the benefit of including recombinant NS1 in the vaccine formulation in animal models. However, these patents and publications do not address or predict a vaccine formulation based solely on E that has demonstrated applicability for human use.
In general the use of non-replicating virus vaccine approaches such as inactivated virus, recombinant subunit protein, and DNA have several advantages over the live-attenuated virus vaccine approaches. Primarily these advantages are related to safety, as no live virus is delivered to subjects, and to the ability to modulate and balance immune responses by adjusting dosage.
In the development of flavivirus vaccines for humans it has been difficult to predict safety and immunogenicity of candidate vaccines in human subjects based on preclinical data in animal models. This has proved challenging for many of the live-attenuated virus vaccine candidates that have advanced to human clinical trials. The most glaring example of a complete failure was the safety profile exhibited by a cloned dengue virus type 3 isolate which displayed a very attractive safety profile in non-human primates, but which induced dengue fever in vaccine recipients in Hong Kong (Sanchez et al., FEMS Immunol. Med. Microbiol. (2006) 24:4914-26). This challenge may be decreased by use of non-replicating virus vaccines which do not require the same level of virus/host interactions in order to achieve vaccine efficacy as replicating virus vaccines. However, there are numerous examples of non-replicating virus vaccine candidates which have shown good safety and protective efficacy in preclinical models, which failed to function as safe and effective vaccines in humans (e.g. inactivated RSV vaccine; Murphy et al., J. Clin. Microbiol. (1986) 24:197-202). Thus, there can be multiple challenges associated to developing safe and effective vaccines for flaviviruses and development often requires years of trial and error. Furthermore, preclinical studies based on animal models may not be predictive of vaccine performance in human subjects; and therefore, human data is critical in demonstrating the utility of a candidate vaccine.
While there are several investigational WNV vaccines in various stages of preclinical research and development, there are only three vaccine candidates that have previously been reported to have advanced to human clinical trials. The three vaccines that have been tested in clinical studies are: (1) a live, attenuated dengue serotype 4-West Nile chimera (Pletnev et al, Proc. Natl. Acad. Sci. USA (2002) 99:3036-41); (2) a live, attenuated Yellow Fever-West Nile chimera (Chimerivax; Monath et al., Proc. Natl. Acad. Sci. USA (2006) 103:6694); and (3) a “naked” DNA vaccine encoding the prM and E genes (Martin et al., J. Infect. Dis. (2007) 196:1732-40). There are intrinsic difficulties and potential shortcomings associated with each of the three candidate vaccines.
The first two vaccines being tested clinically are both live-attenuated vaccines. Safety concerns are paramount with all live viral vaccines given to healthy subjects. Under-attenuation of the virus may result in virus-related adverse events, whereas over-attenuation may abrogate vaccine efficacy. Also, reversion to wild type or mutation to increased virulence (or decreased efficacy) may occur. Moreover, even if properly attenuated, live viral vaccines are contraindicated for specific patient populations, such as immune deficient or immune suppressed patients, as well as particular segments of the normal population, such as pregnant women or elderly individuals. Particularly worrisome with the Chimerivax technology is the safety profile of the YF 17D vaccine (which serves as the backbone for the chimera) in the elderly and immunocompromised. Since the late 1990s a number of cases of fatal, disseminated, viscerotropic and neurotropic vaccine virus infections have been documented, particularly among the elderly and immunocompromised. This has led to recommendations against use of the YF 17D vaccine in these populations unless the risk of YF virus infection is very high (Barrett, ADT and Teuwen, DE (2009) Curr. Opin. Immunol. 21:308-13). In light of the key association between morbidity and mortality of WNV infection in elderly subjects, the application of a live attenuated vaccine approach to this disease target is highly questionable from a safety perspective and thus the first two vaccine candidates do not offer a safe solution to the need for a West Nile vaccine.
The third West Nile vaccine that has been tested in clinical trials is a DNA vaccine. Naked DNA vaccines are unproven for any infectious disease at this time, and the issue of potential immunopathology due to the induction of an autoimmune reaction to the DNA over the long term is unresolved. The results of the WNV DNA vaccine clinical trial were recently reported (Martin et al, J Infect Dis (2007) 196:1732). Low levels of neutralizing antibodies were elicited; however, clinical development of this DNA vaccine has apparently been abandoned, likely linked to safety challenges. Thus, DNA vaccines do not offer a safe and effective solution for development of a WNV vaccine for human use.
As described above, efforts have been made to produce a vaccine that protects humans against disease caused by WNV infection that is both safe and sufficiently immunogenic. Despite these efforts, a WNV vaccine for human use that fully meets these conditions has yet to be established. Therefore, the technical problem to be solved by the invention is the discovery of a WNV vaccine that satisfies two major conditions; the ability to (1) induce relevant protective immune responses in vaccinated individuals (human subjects), and (2) maintain an exceptional safety profile in human subjects in light of the key at-risk population which includes elderly and immunocompromised. This represents a significant challenge in WNV vaccine development, and to date no vaccine approach has been shown to adequately address all aspects of this technical problem. There is an unmet and growing demand, for a solution as the prevalence of West Nile viral infection spreads.